The overall objective of this grant application is to continue to study the mechanism of RNA synthesis of mouse hepatitis virus (MHV), a murine coronavirus, which is of economic and medical importance, and serves as a model for other RNA viruses. During the last grant period, our laboratory has made significant progress toward the understanding of the mechanisms of RNA synthesis, including RNA transcription, replication and recombination. In this grant period, we will concentrate on the following three areas: (l) The study of regulation of (-)-strand RNA synthesis. We will characterize the recognition signals for initiating (-)-strand RNA synthesis, study the kinetics of synthesis and structure of the subgenomic (-)-strand RNA and use temperature-sensitive mutants to study the regulation of synthesis of the various viral RNA species. (2) The study of the trans- and cis-acting sequences required for mRNA transcription. We will characterize the upstream and downstream regulatory sequences for mRNA transcription and study the effects of multiple promoters on RNA transcription. We will also study whether the template for RNA synthesis is a single- or double-stranded RNA, and examine whether transcription can be modulated by a competitor enhancer element. The goal is to determine whether the RNA-dependent RNA transcription in RNA viruses is regulated by a mechanism similar to that in DNA-dependent RNA transcription. (3) The study of the cellular and viral proteins interacting with viral RNAs. We will study the cellular and viral proteins which bind to the 5'- end of (+)-strand and the 3'-end of (-)-strand and the intergenic sequence of MHV RNA. These proteins are likely involved in RNA transcription and replication. We will purify these proteins and characterize their structures, biochemical properties and biological functions. These projects will contribute to our understanding of the mechanism of regulation of RNA synthesis for coronavirus and other RNA viruses.